Interleukin-6-Induced Reciprocal Expression of SERCA and Natriuretic Peptides mRNA in Cultured Rat Ventricular Myocytes

Abstract

We investigated the effect of interleukin-6 (IL-6) expression on sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) mRNA levels in cultured rat neonatal ventricular myocytes. IL-6 plays a key role in regulating cardiac hypertrophy and the development of heart failure, and SERCA, ANP and BNP are all cardiac hormones with regulatory properties. Compared with baseline measurements, treatment with 50 U/ml IL-6 significantly decreased SERCA gene expression, but significantly increased ANP and BNP gene expression in the cardiac myocytes. These results suggest that the clinical overproduction of IL-6 in response to infection, autoimmune disease and cancer might be responsible for cardiac hypertrophy. Cardiac hypertrophy may result from the imbalance of both natriuretic peptides and SERCA transcription levels, caused by elevated IL-6 expression.

Full text

The Journal of International Medical Research
2004; 32: 57 – 61
57
Interleukin-6-induced Reciprocal
Expression of SERCA and Natriuretic
Peptides mRNA in Cultured Rat
Ventricular Myocytes
T TANAKA1, T KANDA2, T TAKAHASHI2, S SAEGUSA2, J MORIYA2AND M KURABAYASHI1
1Second Department of Internal Medicine, Gunma University School of Medicine, Gunma,
Japan; 2Department of General Medicine, Kanazawa Medical University, Ishikawa, Japan
We investigated the effect of interleukin-6
(IL-6) expression on sarco/endoplasmic
reticulum Ca2+-ATPase (SERCA), atrial
natriuretic peptide (ANP) and B-type
natriuretic peptide (BNP) mRNA levels
in cultured rat neonatal ventricular
myocytes. IL-6 plays a key role in
regulating cardiac hypertrophy and the
development of heart failure, and SERCA,
ANP and BNP are all cardiac hormones
with regulatory properties. Compared
with baseline measurements, treatment
with 50 U/ml IL-6 significantly decreased
SERCA gene expression, but significantly
increased ANP and BNP gene expression
in the cardiac myocytes. These results
suggest that the clinical overproduction of
IL-6 in response to infection, autoimmune
disease and cancer might be responsible
for cardiac hypertrophy. Cardiac hyper-
trophy may result from the imbalance of
both
natriuretic peptides and SERCA
transciption
levels, caused by elevated
IL-6 expression.
KEY WORDS: SARCO/ENDOPLASMIC RETICULUM CA2+-ATPASE (SERCA); ATRIAL NATRIURETIC PEPTIDE;
B-TYPE NATRIURETIC PEPTIDE; INTERLEUKIN-6 (IL-6); VENTRICULAR MYOCYTES
Introduction
Sarco/endoplasmic reticulum Ca2+-ATPase
(SERCA) is a major component of beat-to-
beat Ca2+ cycling in the heart. Reduction in
SERCA pump expression and activity has
been associated with diastolic dysfunction in
cardiac hypertrophy1and heart failure.2
Gene transfer of SERCA into adult myocytes
in vitro causes increased contractility of
myocytes and an increased rate of Ca2+
uptake and release.3In contrast, atrial
natriuretic peptide (ANP) and B-type
natriuretic peptide (BNP) are two major
polypeptide hormones that have diuretic,
natriuretic and vaso-relaxant regulatory
properties. They also control growth and pro-
liferation of cells and cardiac hypertrophy.4 – 6
Natriuretic peptides (NPs) are peptide
hormones synthesized in and secreted from
the heart, and play an important role in
cardiovascular homeostasis.7Transgenic
mice overexpressing BNP had reduced blood
pressure and cardiac weight.8
Interleukin-6 (IL-6) is a pro-inflammatory
cytokine with a pivotal role in regulating
cardiac hypertrophy and development of
heart failure.9It is unclear whether IL-6 is a

paracrine hypertrophic factor for cardiac
myocytes, but it may be related to the patho-
physiological status of cardiac hypertrophy.10
Clarification of the relationship between IL-6
and cardiac hypertrophy may lead to
potential treatments for cardiac hypertrophy.
We investigated the effect of IL-6 on
expression of genes involved in regulating
cardiac hypertrophy. The variation in
expression of SERCA, ANP and BNP mRNA in
cultured rat neonatal ventricular myocytes
exposed to different concentrations of IL-6
was measured.
Materials and methods
PRIMARY CULTURES OF RAT
NEONATAL VENTRICULAR
MYOCYTES
Rat neonatal ventricular myocytes were
isolated from 1- or 2-day-old Wistar rats by a
standard method using serial digestion with
tyrosine and collagenase in Ca2+-free Krebs-
Henseleit buffer solution (118 mmol/l
sodium chloride, 4.0 mmol/l potassium
chloride, 1.2 mmol/l magnesium chloride,
1.1 mmol/l KH2PO4, 25 mmol/l sodium
bicarbonate, 5.0 mmol/l glucose and
20 mmol/l N-2-hydroxyethylpiperazine-N'-2-
ethanesulphonic acid, at pH 7.4). After
enriching for myocytes, cells were seeded at a
density of 6 ×105 per 10 cm on a gelatin-
coated culture dish in Dulbecco-modified
minimum essential medium (DMEM)
including 10% fetal calf serum, penicillin
(100 U/ml) and streptomycin (100 µg/ml),
and grown for 48 h. The medium was then
replaced with serum-free DMEM containing
1% ITS (10 µg/ml insulin, 5.5 µg/ml sodium
transferrin, 6.7 ng/ml sodium selenite and
2.0 µg/ml ethanolamine), 0.1% bovine
serum albumin, penicillin and streptomycin.
Using this method we routinely obtained
successful cultures with > 95% rat neonatal
ventricular myocytes. After 48 h of serum-
starvation the cardiac myocytes were
cultured for 6 h in the absence or presence of
IL-6 at concentrations of 1 U/ml, 10 U/ml or
50 U/ml. Endothelin-1 (ET-1; 1 µM) was used
as a control stimulant to activate ANP and
BNP gene expression.
QUANTITATIVE EXPRESSION OF SERCA
AND NATRIURETIC PEPTIDE GENES
Total cellular RNA (20 µg) for northern blot
analysis was prepared from the cardiac
myocyte primary culture using an ISOGEN
kit (Nippon Gene, Toyama, Japan). The RNA
was electrophoresed on a 1.2% agarose/
2.2 M formaldehyde gel and transferred to a
nylon membrane (Hybond-N+, Amersham
International, Tokyo, Japan). Rat-derived
cDNA probes for the SERCA, ANP, BNP and
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) genes were labelled with
[α-32P]dCTP (Amersham International) using
the multi-priming DNA labelling method.
Membranes were pre-hybridized and
hybridized by standard techniques. After
washing with 0.2% sodium citrate
containing sodium chloride (SSC)/0.1%
sodium dodecyl sulphate (SDS) at 42 ºC for
1 h, the blots were exposed to X-ray film at
–80 ºC with intensifying screens. Developed
films were scanned by an image scanner
(ES-800C scanner, Epson America Inc.,
El Segundo, CA, USA) and analysed by
computer program (NIH Image 1.49) to
measure the comparative intensity of each
observed DNA band.
STATISTICAL ANALYSIS
Data are expressed as mean values ± SD. The
association between baseline expression
levels and expression after IL-6 treatment
was analysed by one-tailed analysis of
variance (ANOVA). A P-value < 0.05 was
considered to be statistically significant.
58
T Tanaka, T Kanda, T Takahashi et al.
IL-6-induced expression in cultured myocytes

59
T Tanaka, T Kanda, T Takahashi et al.
IL-6-induced expression in cultured myocytes
FIGURE 1: Interleukin-6 (IL-6)-induced expression of sarco/endoplasmic reticulum
Ca2+-ATPase (SERCA), atrial natriuretic peptide (ANP) and B-type natriuretic peptide
(BNP) mRNA in rat neonatal ventricular myocytes. (A) Total cellular RNA (20 µg), from
cardiac myocytes that were cultured in the absence or presence of IL-6 at
concentrations of 1 U/ml, 10 U/ml and 50 U/ml for 6 h, was analysed by northern
blot. Endothelin-1 (ET-1; 1 µM) was used as a positive control. Glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) mRNA was used to show that comparable
amounts of total RNA were blotted onto the membrane. (B) Comparative expression
of the SERCA, ANP and BNP genes under the influence of IL-6 or ET-1. *P< 0.05 versus
baseline expression levels
1.25
1.00
0.75
0.50
0.25
0
0 1 10 50 ET-1
*
**
*
*
*
*
*
Interleukin-6 (U/ml)
01
10 50 ET-1
Interleukin-6 (U/ml)
ANP
BNP
SERCA
ANP
BNP
SERCA
GAPDH
A
B
Results
Compared with baseline expression, IL-6 sig-
nificantly increased expression of ANP and BNP
mRNA in a dose-dependent fashion (Fig. 1;
P< 0.05). In contrast, IL-6 significantly repressed
expression of SERCA mRNA at concentrations
of 10 U/ml and 50 U/ml (Fig. 1; P< 0.05).

60
T Tanaka, T Kanda, T Takahashi et al.
IL-6-induced expression in cultured myocytes
Discussion
Regulating intracellular calcium concen-
trations through SERCA and other Ca2+
handling proteins leads to control of cardiac
myocyte contractility.11 Impaired expression
and decreased pump activity of SERCA have
been implicated as major components of
cardiac dysfunction. We observed suppressed
SERCA mRNA expression following IL-6
treatment of rat ventricular myocytes.
Sumbilla and colleagues12 described the
efficacy of adenovirus-assisted exogenous
SERCA gene expression in primary cultures of
myocytes from rat neonatal hearts. The
SERCA pump should be assessed as a
therapeutic target for gene therapy of heart
failure.
The natriuretic peptide hormones,
including ANP and BNP, are a family of
vasoactive peptides with many favourable
physiological characteristics. These
hormones have emerged as important
indicators for diagnosing heart failure and
left ventricular dysfunction. Increased
expression of ANP and BNP genes in cardiac
tissue was demonstrated in 25 patients with
mild-to-moderate chronic heart failure
caused by idiopathic dilated cardio-
myopathy.13 Early expression in the heart
of these natriuretic peptides was associated
with disease severity. The ventricular
BNP concentrations were also reported to
increase with the progression of heart
failure.14 Under the stimulation of IL-6
(10 U/ml), early elevated levels of BNP
mRNA expression were found in the cultured
rat ventricular myocytes used in our
experiment. Measuring circulating BNP
concentrations to evaluate prognosis in
patients with cardiac diseases and using
recombinant human BNP to treat heart
failure have been discussed.15
Cultured heart myocytes readily become
responsive to IL-6 stimulation in the
presence of the IL-6 receptor.16 Neighbouring
cells may produce the IL-6 receptor by a
paracrine mechanism in an inflammatory
state, and promote IL-6 action on cardiac
myocytes. This hypothesis has not been
demonstrated in the failing heart, but
evidence from experiments into chronic
inflammatory bowel disease suggests this
may be a potential mechanism.17 Transgenic
mice overexpressing both IL-6 and the
IL-6 receptor had an increased cardiac
weight, with increased left ventricular
wall thickness and cardiomyocyte size.18
In an inflammatory state, therefore,
IL-6 signalling might influence cardiac
hypertrophy through IL-6 receptors in the
myocyte. Further investigations of this
theory are required.
In summary, we found that reciprocal
variations in SERCA, ANP and BNP mRNA
expression in cultured rat neonatal
ventricular myocytes were induced by IL-6
at a concentration of 50 U/ml. These results
suggest that overproduction of IL-6 resulting
from infections, autoimmune diseases and
cancers might be responsible for cardiac
hypertrophy, due to an imbalance of
natriuretic peptides and SERCA expression.
Acknowledgement
This study was supported in part by a grant
from the Science Research Promotion Fund of
the Promotion and Mutual Aid Corporation
for Private Schools of Japan (to TK).
• Received for publication 4 August 2003 • Accepted subject to revision 11 August 2003
• Revised accepted 12 September 2003
Copyright © 2004 Cambridge Medical Publications

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T Tanaka, T Kanda, T Takahashi et al.
IL-6-induced expression in cultured myocytes
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Address for correspondence
Dr T Kanda
Department of General Medicine, Kanazawa Medical University,
1-1, Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa, 920-0293, Japan.
E-mail: kandat@kanazawa-med.u.ac.jp